WO2008021160A2 - Régulation par un nouveau peptide court p53 de la production de cytokines modulée par le facteur litaf - Google Patents

Régulation par un nouveau peptide court p53 de la production de cytokines modulée par le facteur litaf Download PDF

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Publication number
WO2008021160A2
WO2008021160A2 PCT/US2007/017687 US2007017687W WO2008021160A2 WO 2008021160 A2 WO2008021160 A2 WO 2008021160A2 US 2007017687 W US2007017687 W US 2007017687W WO 2008021160 A2 WO2008021160 A2 WO 2008021160A2
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seq
acid sequence
litaf
amino acid
kqsqhmt
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WO2008021160A3 (fr
WO2008021160B1 (fr
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Amar Salomon
Tang Xiaoren
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Boston University
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Boston University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a mutant p53 short peptide useful, for example, in the regulation of the production of cytokines and in the regulation of LPS-induced LITAF and TNF-alpha production.
  • Cytokines are critical players in both the innate and adaptive immune systems. They activate complement, trigger phagocytosis, raise body temperature and activate B and T cells. All of these functions are vital to host defense but cytokine expression must be carefully regulated because these mediators have both favorable and deleterious effects. For example, overproduction of cytokines is thought to contribute to the development of diseases such as diabetes, atherosclerosis and rheumatoid arthritis as well as contribute to inappropriate allergic reactions (Eu, et al, Biochemistry, 39:1375-1384, 2000; Rapoport, et al, Cytokine, 30:219-227, 2005).
  • the p53 protein helps to maintain the genomic stability of cells thereby reducing the probability that a cancer will develop. Loss of p53 function is frequently associated with a broad spectrum of human cancers (Nemoto, S 3 et al, Nutrient availability regulates SIRTl through a fork head-dependent pathway, Science, 306:2105- 2108, 2004).
  • the p53 protein functions as a sequence-specific DNA-binding factor that can activate genes whose promoters contain a p53 response element (Kem, S.E., et al, Identification of p53 as a sequence-specific DNA-binding protein, Science, 252:1708- 1711, 1991; Bode, A.M. and Z.
  • MDM2 induces NF-kB/p65 expression transcriptionally through SpI -binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia, Blood, 99:3367-3375, 2002).
  • LITAF is a transcription factor that is instrumental in the regulation of certain cytokines, either directly or indirectly, especially TNF-alpha, IL-I -beta and VEGF. Inhibition of LITAF mRNA expression resulted in a reduction of TNF-alpha and IL-1-beta expression and an increase in VEGF expression.
  • the binding site for LITAF has been found, for example, within the TNF-alpha promoter region.
  • the regulation of LITAF has also been shown to be critical for the regulation of angiogenesis (provisional application serial no. 60/655,851 which is incorporated herein by reference)
  • the present invention relates to a novel peptide of the septamer sequence KQSQHMT [SEQ ID NO: 1] as well as protein sequences comprising the novel peptide sequence KQSQHMT [SEQ ID NO: I].
  • This sequence comprises amino acids 164 - 170 of the p53 protein.
  • This peptide sequence [SEQ ID NO: 1], and peptides comprising SEQ ID NO: 1, have been identified herein as a regulator of LITAF promoter activity.
  • the sequence KQSQHMT [SEQ ID NO: 1] can be used to modulate the function of the LITAF gene as well as those activates dependent upon the activation of the LITAF gene.
  • Non-limiting examples of those activities include the modulation of the expression of the cytokines'TNF-alpha, IL-1-beta, VEGF 5 IL-2, IL-5, IL-7, IL-12, Eotaxin, IL-15, IFN- ⁇ , IP-IO (human interferon-inducible protein-10), IL-6, RANTES and IL-lra.
  • the present invention relates to the use of the novel peptide sequence KQSQHMT [SEQ ID NO: 1] for the regulation of angiogenesis.
  • the inhibition of LITAF by [SEQ ID NO: 1] and peptides comprising [SEQ ID NO: 1] leads to a decrease in the expression of LITAF.
  • This decrease in available LITAF changes the balance between STAT6B and LITAF in the cell.
  • a relative increase of STAT6B compared to LITAF has been shown to result in an increase in the expression of VEGF which, in turn, modulates angiogenesis (see, U.S. Patent No. 6,566,501 to Amar, et al. and U.S. Patent Provisional Application serial no. 60/655,851 which is incorporated herein by reference).
  • the present invention also relates to nucleic acid sequences that encode the peptide sequence KQSQHMT [SEQ ID NO: I].
  • the present invention also relates to the use of the peptides and nucleotide sequences of the present invention, or derivatives thereof, for the regulation of cytokine expression in organisms, tissues, cells, cell-free systems and diagnostic or assay systems and protocols.
  • the peptides of the present invention or nucleic acid sequences encoding said peptides of the present invention might be transfected into cells, tissues or organisms for the purpose of modulating cytokine activity.
  • Non-limiting examples of the cytokines to be regulated include, for example, TNF-alpha, IL-1-beta, VEGF, IL-2, IL-5, IL-7, IL-12, Eotaxin, IL-15, IFN- ⁇ , IP-10 (human interferon-inducible protein-10), IL-6, RANTES and IL-lra.
  • the present invention relates to the regulation of angiogenesis by the proteins or protein derivatives of the present invention via the regulation of cytokine expression as well as pharmaceutical compositions comprising the proteins or protein derivatives of the present invention.
  • Figure 1 shows the nucleotide sequence of the LITAF promoter.
  • Figure 2 shows a diagram of the LITAF promoter constructs designed for the analysis o,f promoter activities.
  • Figure 3 shows EMSA of the protein-DNA interaction of p53 and the LITAF promoter.
  • Figure 4 (A - C) shows the effects of p53 on LITAF promoter activity in H1299 cells.
  • Figure 5 shows (A) a diagram of major p53 constructs and (B) the analysis of their effects on LITAF promoter activity.
  • Figure 6 shows the effects of transfected p53 constructs on endogenous LITAF gene expression in p53-null H 1299 cells.
  • Figure 7 shows a luciferase assay using p53 peptides.
  • Figure 8 shows the effects of p53 short peptide (pep 12) on LPS- induced endogenous LITAF gene expression in human monocytes.
  • Figures 9A, 9B and 9C show results of a Bioplex ® human monocyte assay.
  • the present invention relates to a novel p53 short peptide [SEQ ID NO: 1] that is involved in the regulation of LPS-induced TNF production as well as compositions derived from the novel peptide and methods for the use of such compositions in modulating cytokine expression in a cell.
  • the present invention is based in part on the identification of a novel peptide (KQSQHMT [SEQ ID NO: I]).
  • KQSQHMT SEQ ID NO: I
  • the novel sequence of the present invention (and derivatives of the sequence) is believed to bind to the LITAF gene promoter sequence thereby reducing and/or inhibiting LITAF gene transcription.
  • the invention provides the use of SEQ ID NO: 1 for modulating the expression of genes other than LITAF. That is, genes that also have a p53 response element or other SEQ ID NO: 1 interactive nucleotide sequence in or near that gene's promoter region.
  • the present invention also relates to the introduction of the short p53 peptide of the present invention into a cell or cells.
  • the novel sequence of the present invention [SEQ ID NO: I] 3 or derivatives thereof, may be introduced into a cell or cells by any of the methods known in the art. Methods of introducing proteins into a cell or cells are well known in 'the art and are provided in detail in, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), which is incorporated herein by reference. In brief, proteins may be introduced into cells in two general ways. The first is with the transfection of nucleic acids expressing the desired protein. Once inside die cell the protein is produced by the cell's own transcription and translation components. The second is with the transfection of the actual protein into the cell.
  • nucleic acid sequence for example, the nucleic acid sequences of the present invention
  • the nucleic acid sequence encoding the desired protein is operably inserted into an expression vector suitable for the transcription and translation of the nucleic acid sequence into the desired protein.
  • a large number of expression vectors are commercially available (e.g., pCAT, Promega, Madison, WI; pBlueScript and pCMV, Stratagene, La Jolla, CA).
  • the splicing of a specific nucleotide sequence into an expression vector is also well know in the art as is referenced above in Sambrook, et al.
  • Suitable transfection methods include DEAE-dextran, calcium phosphate precipitation, LipofectamineTM (Invitrogen, Carlsbad, CA), ProfectinTM (Promega, Madison, WI) and other liposome methods, direct microinjection, electroporation and bioloastic particle delivery, for example.
  • Any primary cell type or any cell line may be used for the present invention.
  • the short p53 peptide of the present invention may also have additional amino acids added to either of the amino or carboxy terminals. Anywhere form zero to 100 or more amino acids may be added to one or both peptide terminals. Examples of sequences to be added may be, for example, tags for the detection of or for determining the location(s) of the peptide within the cell.
  • suitable tethering sequences include the lambda bacteriophage antitermi ⁇ ator protein N (lambdaN-(l-22) or lambdaN peptide) (J. Baron-Benhamou, et al., Methods MoI Biol., 257:135-154, 2004, which is herein incorporated by reference), a glycine-serine tether (US Patent No. 7,074,557, which is herein incorporated by reference).
  • sequences for aiding in the transport of the peptide through, for example, cell or organelle membranes or for transport through selective pores in organelle or cell membranes are well known to those practiced in the art.
  • Transfection of the actual peptides of the present invention may take place by, for example, transport (active or passive) i or by microinjection.
  • Active transport is a process whereby cells absorb material from the outside the cell by engulfing it with the cell membrane. Passive transport may also take place via the passage of peptide fragments, for example, into the cell through pores. Cells frequently transport particles and, especially, proteins and protein fragments into the cell's cytoplasm. Transport may be specific via, for example, specific receptors or it may be more general. With general forms of active transport the cell engulfs constituents from the extracellular milieu. This is often referred to as pinocytosis.
  • Pinocytosis (literally, cell-drinking) is the invagination of the cell membrane to form a pocket filled with extracellular fluid (and molecules within it). The pocket then pinches off to form a vesicle, and the vesicle ruptures to release its contents into the cytoplasm.
  • transfection of the proteins of the present inventon into cells may also be executed by the transfection of nucleic acids that; express the peptide(s) of the preesent invention into cells, as given above.
  • Any primary cell type or any cell line may be used as a recipient for the peptides and nucleic acid sequences of the present invention.
  • the techniques of nucleic acid transfection are . well known in the art (see, for example, Sambrooke, et ah, herein incorporated by reference).
  • Peptides comprising SEQ ID NO: 1 also have the ability to modulate cytokine expression in the methods of the present invention (see, Examples).
  • a biologically active peptide may further be a protein, polypeptide or peptide.
  • protein As defined in this invention, the terms "protein,” “peptide” or “polypeptide” are interchangeable and refer to a sequence of two of more amino acids with or without additional modifications such as, but not limited to, glycosylation.
  • the present invention also relates to methods for the modulation of cytokine expression. These may include, for example, introducing into a cytokine-responsive cell a composition comprising SEQ ID NO: 1.
  • SEQ ID NO: 1 is to be introduced into the cell in an amount effective to modulate cytokine expression. Amounts may vary depending, for example, the target cell or tissue type but can be determined easily via titration of the peptide or expression construct used.
  • the cytokine to be modulated may be, for example, TNF-o. and/or IL- 1/3.
  • Introduction of SEQ ID NO: 1 into the cell decreases expression of TNF-o; and IL- 1/3 and increases expression of VEGF and, as such, this method may be used to effect any of the cellular processes resulting from the same.
  • a biologically active peptide comprising KQSQHMT [SEQ ID NO: 1] may be used in lieu of the septamer peptide of the present invention.
  • a "biologically active peptide” is intended to encompass any mimetic, truncation, deletion and/or substitution or elongation of the peptide sequence of the present invention.
  • the peptide of SEQ ID NO: 1 comprises an additional 0-100 amino acids in sequence with one or both of the amino- or carboxy- terminals of the peptide.
  • the peptide KQSQHMT or proteins comprising the active peptide KQSQHMT may be introduced into a cell by various means in the methods of the present invention.
  • a cell may be contacted directly with the peptide KQSQHMT or proteins comprising active KQSQHMT under conditions for cellular uptake. Such conditions include but are not limited to injection and calcium chloride mediated uptake, electroporation, microinjection, etc.
  • a target cell e.g. , a cytokine-responsive cell
  • a composition comprising KQSQHMT or proteins comprising KQSQHMT may be administered to an animal or individual in a physiologically acceptable carrier in a therapeutically effective amount.
  • Said compound or compounds may be administered alone or in combination with other therapies and may be delivered intravenously, subcutaneously or orally to an animal. Administration may be systemic although local administration may be preferable.
  • the methods of the present invention may be used to study and/or treat diseases associated with aberrant cytokine signaling. It is known in the art that cytokine signaling is involved in pro-inflammatory and anti-inflammatory responses to pathogens and in cellular proliferation and differentiation in a variety of cells. Thus, methods disclosed herein for modulating cytokine signaling may be used to alter these and other cytokine-dependent processes in normal and/or abnormal cells.
  • TNF-alpha tumor necrosis factor- alpha
  • TNF-alpha tumor necrosis factor- alpha
  • Interleukin-1-beta (IL-I -beta) is a cytokine implemented in numerous diseases including acute phase response (the near immidiate response by the immune system in injury that is typically characterized by, e.g., redness and swelling. It is secreted by is secreted by macrophages, monocytes and dendritic cells. The secretion of both TNF-alpha and IL-I -beta are regulated by LITAF expression.
  • the inhibition of LITAF expression by, for example, SEQ ID NO: 1 can be used to modulate the secretion of both these cytokines since decreases in LITAF expression leads to decreases in both TNF-alpha and II- 1 -beta expresion.
  • the modulation of cytokine signaling via the compositions and methods of the present invention may also be used to modulate the immune response of an animal or individual to an antigen, or to treat diseases or repair of damage caused by such as diabetes or inflammatory diseases.
  • compositions and methods for the treatment of nonhealing ulcers for example, in the context of diabetes. Diabetes typically may cause such ulcers ⁇ e.g., tissue damage of the kidney) or be the cause for wounding (e.g., amputation of limbs as a result of disease progression).
  • the compositions and methods of the present invention are ideally suited for the treatment of such conditions via the promotion of angiogenesis.
  • the present invention relates to the interaction of LITAF with a short peptide of the oncogene product p53.
  • Human p53 is a 393-amino acid nuclear transcription factor.
  • the present invention is not limited to any particular theory, it is believed that after binding specifically to the promoter regions of its target genes, p53 activates or inhibits their expression. It is shown in the present invention that p53 directly binds th ⁇ j LITAF promoter region. This binding significantly inhibits LITAF promoter activity and, therefore, suppresses LITAF expression. Therefore, one aspect of the present invention relates to the screening of molecules that may inhibit (or increase) the binding of the p53 short peptide of the present invention to the LITAF promoter.
  • molecules from a library of small molecules are used to form a reaction mixture comprising a small molecule and p53 (or an active fragment thereof, i.e., SEQ ID NO: 1) and a DNA sequence comprising the LITAF promoter region.
  • the mixture is incubated for a length of time and under conditions appropriate for the binding of p53 to the LITAF promoter region.
  • the binding of p53 to the LITAF promoter region is determined (e.g., by electrophoresis or Western blotting).
  • the amount of binding is compared to an identical reaction mixture wherein the small molecule suspected of inhibiting p53 binding to the LITAF promoter region was not added, wherein the decrease in binding in the reaction mixture is indicative of the inhibition of the binding of p53 to the LITAF promoter region.
  • the present invention also relates to compounds and methods for the stimulation of angiogenesis by administering to an organism a suitable amount of the short p53 peptide of the present invention (or an active derivative thereof).
  • Said peptide (or active derivative) may be administered either locally or systemically and may be administered as liquids, pills injections, etc.
  • Suitable excipients may also be added to the identified compound(s) for ease of manufacture, stability, ease of administration, etc.
  • the present invention contemplates the stimulation of angiogenesis by the administration to an organism of a compound capable of inhibiting the transcription of LITAF to the extent necessary to create a measurable stimulation of angiogenesis.
  • Human H1299 a p53- null non-small cell lung cancer cell line (ATCC number: CRL-5803), which contains a homozygous deletion of the p53 gene, was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and adjusted to contain 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate and 10 % FBS.
  • Human monocytes purchased from AB Inc., (Columbia, MD) were grow in RPMI 1640 medium supplemented with 10 % FBS. All human cell cultures were maintained in a 37 0 C humidified atmosphere containing 5 % CO 2 .
  • Plasmid constructs (I) The clone, p53-l, which contains a full length human p53 gene and expresses wild-type p53 protein in mammalian cells, was kindly provided by Dr. Beit Vogelstein (The Johns Hopkins Oncology Center, Baltimore, MD).
  • A12 contained the coordinating amino acids (aa) of p53 (1— 393 ⁇ 101-169);
  • the first mutant DNA fragment was generated by PCR with the primer pair 5'-ATGGAGGAGCCGCAGTCAGAT-S' [SEQ ID NO: 2] and 5'- CCGTCATGTGCTGGGAAGGGACAGAAGATG-S' [SEQ ID NO: 3].
  • the second DNA fragment was generated by PCR with the primer pair 5'- CACATGACGGAGGTTGTGAG-S 1 [SEQ ID NO: 3] and 5'-
  • the p53 site mutant DNA fragment was inserted into the pcDNA3.1/V5-His TOPO vector, (b) Bl contained ⁇ coordinating aa of p53 (l- ⁇ 393 ⁇ 167-234); the first mutant DNA fragment was generated by PCR with the primer pair 5'-ATGGAGGAGCCGCAGTCAGAT-3 l [SEQ ID NO: 7] and 5'-ACATGTAGTTCTGTGACTGCTTGTAGATGG-3 1 [SEQ ID NO: 8]. The second DNA fragment was generated by PCR with the primer pair 5'- AACTACATGTGTAACAGTTCC-3 1 [SEQ ID NO: 9] and 5'-
  • the p53 site mutant DNA fragment was inserted into the pcDNA3.1/V5-His TOPO vector, (c) C6 contained coordinating aa of p53 (1-393 ⁇ 235-300); the first mutant DNA fragment was generated by PCR with the primer pair 5'-ATGGAGGAGCCGCAGTCAGAT-3 1 [SEQ ID NO: 13] and 5 • -TGCTCCCTGGGTAGTGGATGGTGGTACAGT-3 1 [SEQ ID NO: 14]. The second DNA fragment was generated by PCR with the primer pair 5'- CCAGGGAGC ACT AAGCG AGCA-3 1 [SEQ ID NO: 15] and 5'- TCAGTCTGAGTCAGGCCCT-S' [SEQ ID NO: 16].
  • Both the first and second PCR- generated DNA fragments were purified and diluted as template to 1 ng per reaction, and amplified by PCR with the primer pair 5'-ATGGAGGAGCCGCAGTCAGAT-S ' [SEQ ID NO: 17] and 5 1 -TCAGTCTGAGTCAGGCCCT-3 1 [SEQ ID NO: 18].
  • DlO contained coordinating aa of p53 (1-393 ⁇ 156-178);
  • the first mutant DNA fragment was generated by PCR with the primer pair 5'-ATGGAGGAGCCGCAGTCAGAT-3 l [SEQ ID NO: 19] and S'-GGTGCCGGGCGGGGGTGTGGA-S' [SEQ ID NO: 20].
  • the second DNA fragment was generated by PCR with the primer pair 5'-CATGAGCGCTGCTCA-3' [SEQ ID NO: 21] and 5 l -TCAGTCTGAGTCAGGCCCT-3 • [SEQ ID NO: 22].
  • Both the first and second PCR-generated DNA fragments were purified and diluted as template to 1 ng per reaction, and amplified by PCR with the primer pair 5'- ATGGAGGAGCCGCAGTCAGAT-3' [SEQ ID NO: 23] and 5'-
  • p53 site mutant DNA fragment was inserted into the pcDNA3.1 /V5-His TOPO vector, (e) p53LFB 12 contained coordinating aa of p53 from 164 to 170 but added one methionine (M) for initiation; the DNA fragment with the primer pair 5'- ATGAAGCAGTCACAGCACATGACGA-3' [SEQ ID NO: 25] and 5'- CGTCATGTGCTGTGACTGCTTCATA-S' [SEQ ID NO: 26] was annealed by heating for 2 minutes at 65°C, then cooling slowly to ⁇ 35°C over 15-30 minutes.
  • the annealed DNA fragment was inserted into the pcDNA3.1/V5-His TOPO vector, (f) 12REV contained the coordinating reverse aa of p53 LFB 12 above from 170 to 164 as a control, but added one methionine (M) for initiation; the DNA fragment with the primer pair 5'- ATGGCAGTACACGACACTGACGAAA-3' [SEQ ID NO: 27] and 5'- TTCGTC AGTGTCGTGTACTGCC ATA-3' [SEQ ID NO: 28] was annealed by heating for 2 minutes at 65 0 C, then cooling slowly to ⁇ 35°C over 15-30 minutes. The annealed DNA fragment was directly inserted into the pcDNA3.1/V5-His TOPO vector.
  • the full length LITAF promoter DNA was generated from human genomic DNA by PCR with the primer pairs: 5'- CCAGAGGGCCGGGAGCGCCCCA-B 1 [SEQ ID NO: 29] and 5'- TTTACCCAGCACCGGCGGTGGA-3' [SEQ ID NO: 30], and was subcloned into a vector, pGL3-basic (Promega).
  • LITAF promoter DNA was generated by PCR using pGLP990 as the template, using the following primer pairs: (a) 5'- CGGGGAACCGGCG ATGGTCTC-3' and 5'-TTTACCCAGCACCGGCGGTGGA-S' [SEQ ID NO: 31] for the region -700-+100 of the LITAF promoter; (b) 5'- GCCCCCGCCCCCGTCCCCGCC-3' [SEQ IDNO: 32]and 5'-
  • TTTACCCAGCACCGGCGGTGGA-S 1 for the region -600-1-100 of the LITAF promoter; (c) 5'-GGCCAGCTCAGACCTCCCGGC-S' [SEQ ID NO: 34] and 5'-TTTACCCAGCACCGGCGGTGGA-3 [SEQ IDNO: 35] for -550-H00'; (d) 5'- CGGCGCGGGGACGCCGGGGCG-3' [SEQ ID NO: 36] and 5'- TTTACCCAGCACCGGCGGTGGA-S' [SEQ ID NO: 37] for-500 ⁇ +100; (e) 5'- GGTGGCGCCAGCACCTGCTGG-3' [SEQ ID NO: 38] and 5'- TTTACCCAGCACCGGCGGTGGA-S' [SEQ ID NO: 39] for -450-+100; (f) 5'- GCCTCCTGGGATGCCAGGGGG-3' [SEQ ID NO: 40] and 5'-
  • DNA probes DNA without mutation was amplified from pGLP990 DNA as the template by PCR using the primer pairs 5'-GCCCCCGCCCCCGTCCCCGCC-S' [SEQ ID NO: 42] and 5'-CCCCAGCCAAGGGCTCAGTGC-S ' [SEQ ID NO: 43] for the region -600 — 400 of LITAF promoter; the primer pairs 5 '- GCCCCCGCCCCCGTCCCCGCC-3' [SEQ ID NO: 44] and 5'-
  • CTGGCGCCACCGGCCCCCCGC-3' [SEQ ID NO: 45] were used for -600—440; and the primer pairs 5'-GCGCCCGAGAGGCCAGCTCAGA-S' [SEQ ID NO: 46] and 5'- CCCCAGCCAAGGGCTCAGTGC-3' [SEQ ID NO: 47] were used for -560—400.
  • DNA containing deletions was generated as follows. The double-stranded oligonucleotide (ds- oligo) was annealed by heating for 2 minutes at 65.C, then cooled slowly to ⁇ 35.C over 15-30 minutes with the follow primer pairs: 1) 5'-
  • each 0.01 ⁇ g annealed double strand (ds)-oligo was amplified without additional primers or template by PCR.
  • the DNAs were purified using a gel extraction kit, (Qiagen) and each DNA species was labeled with [ ⁇ 32 P]ATP using T4 polynucleotide kinase (Promega) following the manufacturer's instructions.
  • Labeled double-stranded DNA oligos were purified using G-25 Sephadex columns (Boehringer) and precipitated with ethanol. After centrifugation, the DNA pellets were suspended in 10 ⁇ l water and aliquots of each oligo were measured for cpm/ ⁇ l and used as probes for EMSA, as described below.
  • Electrophoresis mobility shift assay U 2 OS cells were used for EMSA due to its high DNA transfection efficiency.
  • the cell cultures (5 x 10 6 ) were transfected with 1 ⁇ g of p53-l DNA using Lipofectamine 2000 (Invitrogen) for 3 hrs, washed with PBS, then incubated overnight in medium (DMEM + 10 % FBS) at 37 0 C, in a humidified atmosphere containing 5 % CO 2 -
  • the proteins from cells were extracted with lysis buffer (Promega) plus a cocktail of protein inhibitors (Sigma) following the manufacturer's instruction. The proteins were used for EMSA.
  • a reaction mixture contained' 10 ⁇ g of extract proteins, 1 x 10 5 cpm/ ⁇ l radiolabeled double-stranded oligo DNA probe, 3 ⁇ g of poly dl-dC (deoxyinosinic acid deoxycytidylic acid; Sigma), 5 ⁇ g of BSA, 4 ⁇ l of gel shift binding 5* buffer (Promega), and nuclease-free water to achieve a final volume of 20 ⁇ l. Mixtures were incubated at room temperature for 30 min, followed by electrophoresis on non-denaturing 6 % polyacrylamide gels in Tris-borate-EDTA buffer (90 mM Tris-borate/2 mM EDTA HEPES, pH 8.0).
  • the proteins from the treated cells were extracted with lysis buffer (Promega) plus a cocktail of protein inhibitors (Sigma) following the manufacturer's instructions and suspended in SDS sample buffer, heated at 95°C for 5 min, then applied to SDS-polyacrylamide gels and detected by Western blotting.
  • Antibodies were purchased from the following vendors: LITAF (611615, BD Biosciences), actin (C-11, Santa Cruz), p53 (FL-393, Santa Cruz) or luciferase (NB 600-307, Novus Bio. Inc).
  • Luciferase assay Cultures OfU 2 OS (5 x 10 6 ), p53-null human H1299 non- small cell lung cancer cells (5 x 10 6 ) or p53 wildtype human monocytes (1 * 10 6 ) were co-transfected with DNAs using Lipofectamine 2000 (Invitrogen) for 3 hrs, washed with PBS, then further treated with peptides by Chariot kit (Active Motif, Carlsbad, CA) as required. Cells were then incubated overnight in appropriate media (DMEM or RPMI 1640) + 1.0 % FBS at 37 0 C, in a humidified atmosphere containing 5 % CO 2 .
  • appropriate media DMEM or RPMI 1640
  • ⁇ - galactosidase gene was included in all transfections. Cells were harvested and the luciferase activity of each lysate was measured (Turner Designs luminometer model TD- 20/20) using a commercial kit (luciferase reporter assay system, Promega) according to the protocol provided by the manufacturer. Finally, the data were normalized to ⁇ - galactosidase expression. [0055] ELISA. Human monocytes (1 x 10 6 ) were stimulated with 0.1 ⁇ g/ml E. coli LPS for 3 hrs, washed with PBS, then further treated with peptides by Chariot kit (Active Motif, Carlsbad, CA).
  • LITAF is linked to the well known sensor and mediator of damage-induced apoptosis, p53 (Polyak, et aL, Nature, 389:300-305, 1997, which is herein incorporated by reference).
  • Human p53 is a 393-amino acid nuclear transcription factor. After binding specifically to the promoter regions of its target genes, p53 activates their expression. This example determined the nature of the signaling involved in p53 accumulation and LITAF mRNA induction after LPS stimulation. It was demonstrated that a direct binding between p53 and LITAF promoter took place. The region on the LITAF promoter where p53 binds was also identified. The specific site required for p53 binding is located between -600 to -480bp on the LITAF promoter and binding of p53 protein significantly inhibits LITAF promoter activity.
  • Figure 1 the sequence lacking a TATA box but including the presence of multiple GC boxes was indicated ( Figure 1).
  • the transcription start site is indicated by a +1 as described (Myokai, F., et al., A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization and chromosomal assignment, Proc. Natl. Acad. ScL USA, 96:4518- 4523, 199.9).
  • the first amino acid of the ORF is indicated by a rectangle. Underlines represent GC bases.
  • the punitive site for p53 binding from -550 to -500 is surrounded with a box.
  • deletion promoter constructs were generated containing the 1,090 bp promoter vector (-990 to +100) and a series of 5' upstream sequence deletions. Each of these constructs, the full- length luciferase reporter construct pGLP990 and deletion constructs pGLP700, pGLP600, pGLP550, pGLP500, pGLP450 and pGLP400 ( Figure 2), were individually transfected into U 2 OS cells.
  • LITAF promoter 1 DNA was fused to a promoter-less luciferase reporter vector, pGL3-basic.
  • the open box represents the LITAF promoter and the filled box represents the luciferase reported gene (Luc).
  • the full-length construct containing a sequence from the region - 990 bp to +100 bp of LITAF promoter yielded the highest luciferase activity, which was set at 100 % for comparison with deletion constructs. Triplicate assays were performed. Values were normalized by ⁇ -gal assay.
  • the protein extract from the pGLP990-transfected cells was assigned the maximal promoter activity value of 100 %.
  • the relative activity that could be detected decreased gradually: pGLP700 (98 %), pGLP ⁇ OO (96 %), pGLP550 (83 %), pGLP500 (34 %), pGLP450 (3.5 %) and pGLP400 (2.8%).
  • the results showed that the element required for self-transactivation activity was mainly located in the region upstream to -500 as indicated by the open bars in Figure 2.
  • p53 Binding Activity To analyze binding of p53 to the LITAF promoter sequence, EMSA was performed with the protein extract from p53-l-transfected (p53-l is wild type p53) U 2 SO cells (a human bone osteosarcoma epithelial cell line), ( Figure 3, lanes 3 - 8) in the appropriate buffer with one DNA probe (-600 ⁇ - 400, -600 ⁇ - 440, -550 ⁇ - 40O 5 -600 ⁇ - 400 ⁇ - 440 ⁇ - 440, -600 ⁇ - 400 ⁇ -520 ⁇ - 480 or -600 ⁇ - 400 ⁇ -560 ⁇ -520; see, infra) in separate lanes.
  • Lane 2 also contained a 50-fold excess of unlabeled competitor.
  • the DNA amplified from a different region of the LITAF promoter was labeled with [ 32 P] ATP (Tarig, et al., Identification and functional characterization of a novel binding site on TNF-alpha promoter, Proc. Natl. Acad.
  • p53- null H1299 cells jp ⁇ S ' ⁇ were co-transfected with pGLP700 and p53-l and then protein extracts were analyzed by Western blot.
  • p53-null H1299 cells were co-transfected with 0.5 ⁇ g of pGLP700, pGLKP ⁇ OO, pGLP550, pGLP500, pGLP450 or pGLP400, plus 0.5 ⁇ g of p53-l.
  • the concentration of luciferase from each test was individually measured by a luciferase reporter system (Promega, Madison, WI). Triplicate assays were performed. Values were normalized to ⁇ -gal production. The figure compares effects of p53 on promoter activity in cells containing p53 (filled holes) or lacking p53 (open box).
  • Deletions, indicated by ⁇ correspond to aa 101 - 167 in construct A12, aa 167 - 234 in Bl, aa 235 - 300 in
  • Construct p53LFB12 contained only 7 aa (KQSQHMT [SEQ ID NO: I]) from the region (aa 164 - 170).
  • the LITAF promoter activity was strongly reduced to 9 % of control due to transient transfection of wild-type p53 (p53-l).
  • the proteins extracted from each transfection of construct plus pGLP700 compared to the transfection of pGLP700 alone in p53-null H1299 cells were measured by luciferase assay. Triplicate assays were performed. The results showed partial LITAF promoter activity was maintained by the expression of wither Al 2 (73 %) or Bl (61 %), which lacked aa 101- 167 or aa 167-234, respectively.
  • Construct C6 containing aa 167 and its flanking sequences, reduced LITAF promoter activity to 22 % of control even though it lacked aa 235-300. This suggested that the specific residues playing an important role in the inhibition of LITAF promoter activity were located in the region around aa 167.
  • the DNA clones DlO and p53LFB12 which either possessed (DlO) or lacked (p53LFB12) aa 167 and its flanking sequences were constructed.
  • Construct DlO lacking aa 156-178 completely lost its ability to inhibit LITAF promoter activity.
  • pep 12 short peptides (pep 12) that contain 7 residues (aa 164 - 170) (KQSQHMT [SEQ ID NO: I]) corresponding to p53LFB12 and another peptide (pepSC) with reverse sequence of pep 12 as a negative control, were synthesized and analyzed using the luciferase assay.
  • p53-null H1299 cells (5 x 10 6 ) were transfected with pGLP700 (# 2 - 4, # 7 - 12), pGLP550 (# 13 and 14) or pGLP500 ( # 15 and 16) plus p53LFB12 (# 4) or plus 12REV (# 3) as control using Lipofactamine 2000 (Invitrogen) for 3 hours, washed with PBS and further transfected with peptide, 10 ⁇ g/ml (# 10, 12, 14 and 16), then cells were incubated in appropriate medium at 37 0 C overnight. Luciferase activities of the lysates from each culture of treated cells were measured.
  • Example 8 [0086] Analysis of p53 Short Peptide- mediated LPS-induced LITAF of Human TNF- ⁇ Production in Human Monocytes
  • the cells were incubated in appropriate medium at 37 0 C, 5 % CO2 overnight.
  • the supernatants from cell cultures were measured by ELISA (Abraxis, Warminster, PA) and the extracts were detected by Western blot with antibodies against LITAF and actin (as a control).
  • This assay was performed to determine the effectiveness of the peptide of the present invention [pepl2: SEQ ID NO.: 1] to inhibit various human cytokines.
  • the assay performed was a human monocyte assay well known to those practiced in the art.
  • the kit used here (trade name Bioplex) was supplied by Bio-Rad (Hercules, CA). Other commercially available assays are known and suitable for use in the assay described here.
  • % Results are shown in Figures 9 A, 9B and 9C.
  • Bar (1) is the carrier solution alone (chariot alone);
  • bar (2) is E. coli LPS stimulant;
  • bar (3) isi?. coli LPS + pepSC (scramble peptide);
  • bar (4) is E. coli LPS + pepl2 (10 ⁇ g);
  • bar (5) is E. coli LPS + pepl2 (100 ⁇ g).
  • the cytokines tested include Human (Hu) IL-2, Hu 11-5, Hu 11-7, Hu 11-12 Hu Eotaxin, Hu 11-13, Hu-PDGF- ⁇ , Hu IL-l ⁇ , Hu-15, Hu IFN- ⁇ , Hu IP-10, Hu IL-6, Hu RANTES, Hu TNF- ⁇ , Hu Il-lra and Hu VEGF. Most of the cytokines tested showed significant reduction in the concentration of active cytokine in the assay with the ! exception ' s of Hu IL-13, Hu PDGF- ⁇ and Hu RANTES. These cytokines showed only minor reductions in active cytokines.

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Abstract

La présente invention concerne un nouveau peptide isolé et purifié ayant la séquence KQSQHMT [SEQ ID n° : 1] ; des séquences d'acides nucléiques codant pour ladite séquence de peptide et capable d'exprimer ladite séquence sous forme d'une protéine exogène dans une cellule cible ; ainsi que des procédés pour la réduction ou l'inhibition de l'activité du facteur LITAF en transfectant dans une cellule cible le peptide KQSQHMT [SEQ ID n° : 1] ou un acide nucléique capable d'exprimer ledit peptide. La présente invention concerne également l'utilisation du nouveau peptide (et des séquences de nucléotides correspondantes) de la présente invention pour la régulation de l'expression de cytokines dans des cellules cibles. La présente invention concerne également l'utilisation du nouveau peptide (et des séquences de nucléotides correspondantes) de la présente invention pour la régulation de réponses inflammatoires chez des mammifères.
PCT/US2007/017687 2006-08-17 2007-08-09 Régulation par un nouveau peptide court p53 de la production de cytokines modulée par le facteur litaf Ceased WO2008021160A2 (fr)

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